Angiogenesis is the process by which new blood vessels form. In response to specific chemical signals, capillaries sprout from existing vessels, eventually growing in size as needed by the organism. Initially, endothelial cells, which line the blood vessels, divide in a direction orthogonal to the existing vessel, forming a solid sprout. Adjacent endothelial cells then form large vacuoles and the cells rearrange so that the vacuoles orient themselves end to end and eventually merge to form the lumen of a new capillary (tube formation).
Angiogenesis is stimulated by a number of conditions, such as in response to a wound, and accompanies virtually all tissue growth in vertebrate organisms such as mammals. Angiogenesis also plays a role in certain disease states such as diabetic retinopathy and certain cancers. The growth of tumors, for example, requires blood vessel growth to provide oxygen and nutrients to the growing tumor tissue.
Angiogenesis may be arrested or inhibited by interfering with the chemical signals that stimulate the angiogenic process. For example, angiogenic endothelial cells produce proteases to digest the basal lamina that surround the blood vessels, thus clearing a path for the new capillary. Inhibition of these proteases, or their formation, can prevent new vessels from forming. Likewise, the endothelial cells proliferate in response to chemical signals. Particularly important proliferation signals include the vascular endothelial growth factor (VEGF), and the fibroblast growth factor (FGF) families of proteins. VEGF has been shown to be involved in vascularization of certain tumors. Interference with these proliferation signaling processes can also inhibit angiogenesis.
Kiosses et al. J. Cell Biol., 1999, 147:831–843 (hereinafter “Kiosses et al.”), have identified the protein kinase p65 PAK1 (PAK-1) as a potential modulator of endothelial cell migration. PAK-1 is a 65 kD serine/threonine kinase that serves as a direct effector of Rac and Cdc42. The downstream function of PAK-1 is complex, but it is clearly implicated in regulation of the actin cytoskeleton and cell migration. Kiosses, et al. also reported that expression of dominant negative PAK-1 strongly inhibited growth factor-induced endothelial cell movement. The inhibition appeared to be mediated by a decrease in contractility, and it was suggested that PAK-1 plays a role in myosin-dependent contraction and detachment of the rear of the cell. This study also mapped the inhibitory effect of dominant negative PAK-1 to a single, 74 amino acid construct having a 12 amino acid, proline-rich sequence near the amino-terminus of the protein. This sequence was reported by Lu et al., Curr. Biol., 1996, 7:85–94, to bind the SH3 domain from the adapter protein Nck and has been proposed to target PAK-1 to the cell membrane via an interaction with Nck.
There is an ongoing need for effective methods of inhibiting angiogenesis. The present invention provides a method of inhibiting angiogenesis in a vertebrate organism using proline-rich oligopeptides, which are synthetic polypeptides containing proline-rich amino acid sequences that specifically target proliferating endothelial cells, inhibiting cell migration processes that are necessary for blood vessel formation.